1. Field of the Invention
The invention relates to the use of natural product compounds isolated from plants, and synthetic chemical analogues thereof, for the prevention and treatment of beta-Amyloid protein-induced disease. Specifically, the invention relates to pharmaceutical compositions that protect neuronal cells from beta-Amyloid insult for use in preventing and treating beta-Amyloid protein-induced disease. More particularly the invention is directed to protection of retinal cells and other visual function related cells of the eyes from beta-amyloid involved pathology, in particular, glaucoma and age-related macular degeneration (AMD).
2. Description of Related Technology
Alzheimer's disease (AD) is the most common cause of progressive cognitive dysfunction. AD affects approximately four million Americans and causes more than 100,000 deaths each year, with a total annual cost approaching $100 billion. It is estimated that by the year 2020, 14 million Americans will be afflicted by the disease. See Carr et al., Am J Med 103, 3S (1997) and Shastry, Am J Med Sci 315, 266 (1998). Furthermore, AD has a profound effect on the millions of family members and other loved ones who provide most of the care for people having this disease. Unfortunately, the cure for AD has not yet been discovered.
The principal pathological characteristics of AD are senile plaques and neurofibrillary tangles (NTFs). Senile plaques are extracellular deposits principally composed of insoluble aggregates of beta-amyloid (βA), that are infiltrated by reactive microglia and astrocytes. See Seidl et al., Neurosci. Lett 232, 49 (1997), Yan et al., Nature 382, 685 (1997), Goedert, Trends Neurosci 16, 460 (1993), Haass et al., Cell 7, 1039 (1994), Trojanowski et al., Am J Pathol 144, 449 (1994), Davis et al., Biochem Biophys Res Commun 189, 1096 (1992), Pike et al., Neuroscience 13, 1676 (1993), Hensley et al., Proc Natl Acad Sci USA 91, 3270 (1994), Behl et al., Cell 77, 817 (1994), Meda et al., Nature 374, 647 (1995), and Klegeris et al., Biochem Biophys Res Commun 199, 984 (1994). Plaques are diffusely distributed throughout the cerebral cortex of AD patients, and are the neuropathologic hallmark of the disease. See Seidl et al., Neurosci Lett 232, 49 (1997), Yan et al., Nature 382, 685 (1997), Goedert, Trends Neurosci 16, 460 (1993), Haass et al., Cell 7, 1039 (1994) and Trojanowski et al., Am J Pathol 144, 449 (1994). These plaques or βA fibril deposits are believed to be responsible for the pathology of a number of neurodegenerative diseases including, but not limited to, Alzheimer's disease. NTFs are intraneuronal accumulation of paired helical filaments composed mainly of an abnormal form of tau protein, that is a microtubule associated phosphoprotein which can promote microtubule formation. See Goedert, Trends Neurosci 16, 460 (1993), Haass et al., Cell 7, 1039 (1994) and Trojanowski et al., Am J Pathol 144, 449 (1994). In the AD brain, the tau protein in NFTs is hyperphosphorylated (See Ihara et al., J Biochem 99, 1807 (1986)), a condition which has been suggested to contribute to the destabilization of microtubule network, thereby impairing axonal network, and eventually causing neuronal death. See Trojanowski et al., FASEB J 9, 1570 (1995). NTFs occur primarily in medial temporal lobe structures (hippocampus, entorhinal cortex, and amygdala), and NTFs density appears to correlate with dementia severity.
Senile plaques and NTFs appear to be involved in cerebral amyloid angiopathy, consequent neuronal loss, and cerebral atrophy leading to dementia. Although research findings suggest that both plaques and NTFs are involved in disrupting nerve cell functions, the mechanisms that lead to the pathology are not clearly understood.
βA has been suggested as one of the major causes of AD. βA was shown to exert direct toxic effects on neurons and to inhibit neurite growth in vitro in a dose dependent manner. Thus, therapeutic approaches that can modulate βA toxicity have been hypothesized to represent important methods for controlling the onset of AD. It is envisioned that if neuronal cells can be protected from βA/senile plaque-induced toxicity, the onset of AD may be delayed or prevented. Current pharmacological approaches related to AD preventive and neuroprotective interventions include antioxidant therapy (See Lucca et al., Brain Res 764, 293 (1997), Pike et al., J Neurochem 69, 1601 (1997), Manelli et al., Brain Res Bull 38, 569 (1995), Parnetti et al., Drugs 53, 752 (1997), Zhou et al., J Neurochem 67, 1419 (1996), Kumar et al., Int J Neurosci 79, 185 (1994), Preston et al., Neurosci Lett 242, 105 (1998), and Tatton et al., Neurology 47, S171 (1996)), acetylcholinesterase inhibitors (See Hoshi et al., J Biol Chem 272, 2038 (1997), Maurice et al., Brain Res 706, 181 (1996), Harkany et al., Brain Res 695, 71 (1995), and Lahiri et al., J Neurosci Res 37, 777 (1994)), nicotinic and muscarinic agonists (See Maurice et al., Brain Res 706, 181 (1996), and Kihara et al., Brain Res 792, 331 (1998)), estrogen (See Ihara et al., J Biochem 99, 1807 (1986), Henderson, Neurology 48 (5 Suppl. 7), S27 (1997), and Green et al., Neuroscience 84, 7 (1998)), nerve growth factor (NGF) (See Hefti, Neurobiol Aging 15 (Suppl 2), S193 (1994), and Seiger et al., Behav Brain Res 57, 255 (1993)), calcium channel blockers (See Zhou et al., J Neurochem 67, 1419 (1996) and Friedlich et al., Neurobiol Aging 15, 443 (1994)), Zinc (See Cuajungco et al., Neurobiol Dis 4, 137 (1997)), sulfonated compounds (See Pollack et al., Neurosci Lett 197 211 (1995) and Lorenzo, et al., Ann NY Acad Sci 777, 89 (1996)), triaminopyridine nonopiate analgesic drug (See Muller et al., J Neurochem 68, 2371 (1997)), low molecular lipophilic compounds that can activate neurotrophic factor signaling pathway (See Mattson, Neurosci Biobehav Rev 21, 193 (1997)), and non-steroidal anti-inflammatory drugs such as ibuprofen and aspirin (See Parnetti et al., Drugs 53, 752 (1997), Beard et al., Mayo Clin Proc 73, 951 (1998), and Pasinetti et al., Neuroscience 87, 319 (1998)). Of particular interest to the present invention is the observation that an anti-βA protein antibody was shown to clear senile plaques and protect mutant PDAPP mice from the onset of AD. See St George-Hyslop et al., Nature 400, 116 (1999).
The generation of reactive oxygen intermediates (ROS) through oxidative stress caused by βA has been suggested to be the major pathway of the βA-induced cytotoxicity. See Klegeris et al., Biochem Biophys Res Comun 199, 984 (1994) and Lucca et al., Brain Res 764, 293 (1997). Senile plaques have been shown to exert a cytotoxic effect on neurons by stimulating microglia to produce reactive oxygen species (ROS). See Seidl et al., Neurosci Lett 232, 49 (1997), Yan et al., Nature 382, 685 (1997), Goedert, Trends Neurosci 16, 460 (1993), Haass et al., Cell 7, 1039 (1994), Trojanowski et al., Am J Pathol 114, 449 (1994), Davis et al., Biochem Biophys Res Commun 189, 1096 (1992), Pike et al., Neuroscience 13, 1676 (1993), Hensley et al., Proc Natl Acad Sci USA 91, 3270 (1994), Behl et al., Cell 77, 817 (1994), Meda et al., Nature 374, 647 (1995) and Klegeris et al., Biochem Biophys Res Commun 199, 984 (1994). The damaging effect of ROS can be prevented by the free radical scavenging enzyme superoxide dismutase (SOD). See Thomas et al., Nature 380, 168 (1996) and Manelli et al., Brain Res Bull 38, 569 (1995).
Aging of synthetic βA for 7 to 14 days at 37° C. in modified Eagle's media was also demonstrated to cause neurotoxic free radical formation. See Friedlich et al., Neurobiol Aging 15, 443 (1994) and Puttfarcken et al., Exp Neurol 138, 73 (1996). However, aging PA in the presence of the media supplement B27, which contains antioxidants as well as other agents that provide protection against oxidative damage, has been shown to inhibited βA-induced neurotoxicity. See Thomas et al., Nature 380, 168 (1996) and Manelli et al., Brain Res Bull 38, 569 (1995).
In addition to βA peptide-induced ROS mediated neurotoxicity, βA peptide has been shown to cause neuronal cell death by stimulating microglial expression of tumor necrosis factor β (TNFβ). See Tarkowski et al., Neurology 54, 2077 (2000) and Barger et al., Proc Natl Acad Sci USA 92, 9328 (1995). The accumulation of βA peptide as neuritic plaques is known to be both trophic and toxic to hippocampal neurons, causing apoptosis or necrosis of the neurons in a dose dependent manner. βA peptide was demonstrated to induce these cellular effects by binding with a receptor for advanced glycation end products (RAGE) that was previously known as a central cellular receptor for advanced glycation endproducts. See Arancio et al., EMBO J23, 4096 (2004), Huttunen et al., J Biol Chem 274, 19919 (1999), and Yan et al., Nature 382, 685 (1996). RAGE was suggested to mediate the interaction of PA peptide with neurons and with microglia, resulting in oxidative stress mediated cytotoxicity. Blocking RAGE with anti-RAGE F(αβ′)2 prevented the appearance of TNFβ messenger RNA and diminished TNFβ antigen to levels seen in untreated cells. Thus, it is postulated that RAGE mediates microglial activation by βA peptide by producing cytotoxic cytokines that cause neuronal damage in AD patients. In addition, RAGE was also demonstrated to specifically bind with βA peptide and mediate βA peptide-induced oxidative stress.
Cell receptors that bind to βA peptide have been identified. The low-affinity neurotrophin receptor p75 (p75NTR) which belongs to the family of apoptotic receptors that generate cell-death signals on activation was found throughout the brains of AD patients. βA peptide was found to be a ligand for p75NTR, and to cause preferential apoptosis of neurons and normal neural crest-derived melanocytes that express p75NTR upon specifically binding to p75NTR. See Zhang et al., J Neurosci 23, 7385 (2003) and Perini et al., J Exp Med 195, 907 (2002).
Basal forebrain cholinergic neurons express the highest levels of p75NTR in the adult human brain and have been shown to be involved in AD. The expression of p75NTR neuronal cells was shown to potentiate βA peptide-induced cell death. This interaction of βA peptide with p75NTR to mediate neuronal death in AD suggested a new target for therapeutic intervention. See Zhang et al., J Neurosci 23, 7385 (2003) and Perini et al., J Exp Med 195, 907 (2002).
Recently, ERAB which is over-expressed in neurons of the AD brain, was shown to bind with βA peptide to induce neuronal death in AD. Blocking ERAB with an antibody, anti-ERAB F(ab′)2, was found to reduce the βA peptide-induced cell death while ERAB overexpression increases βA peptide-induced cell death. See Frackowiak et al., Brain Res 907, 44 (2001) and Yan et al., J Biol Chem 274, 2145 (1999).
In designing inhibitors of βA peptide toxicity, it was found that neither the alteration of the apparent secondary structure of βA peptide nor the prevention of βA peptide aggregation is required to abrogate the cytotoxicity of βA peptide. Nonetheless, inducing changes in aggregation kinetics and in higher order structural characteristics of βA peptide aggregates also proved to be effective in reducing βA peptide toxicity. See Soto et al., Neuroreport 7, 721 (1996). Synthetic inhibitors that interact with βA peptide were shown to completely block βA peptide toxicity against PC12 cells, demonstrating that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity. It was also demonstrated that dipolar compounds such as phloretin and exifone that decrease the effective negative charge of membranes can prevent the association of βA peptide with negatively charged lipid vesicles and thereby prevent βA peptide-induced cytotoxicity. See Hertel et al., Proc. Natl. Acad. Sci. USA 94, 9412 (1997). These results suggest that PA peptide toxicity can be mediated through a physicochemical interaction with cell membranes.
Glaucoma and age-related macular degeneration (AMD) are the most common leading cause of irreversible progressive visual dysfunction that leads to blindness. Glaucoma causes irreversible vision loss worldwide an estimated 66.8 million people. See Khaw et al., BMJ 320, 1619 (2000). AMD is the leading cause of blindness and vision loss in developing countries due to increased life expectancy and subsequent increase in aged population. See VanNewkirk et al., Ophthalmol 108, 960 (2001). Between 20 and 25 million people are affected by AMD worldwide, a figure that will triple with the increase in the aging population in the next 30˜40 years. See Smith et al., Ophthalmol 108, 697 (2001) and McCarty et al., Arch Ophthalmol 119, 1455 (2001). There are over 200,000 cases of neovascular degeneration that present to ophthalmologist in the United States each year. See Bressler et al., BMJ 321, 1425 (2000) and Chopdar et al., BMJ 326, 485 (2003). Glaucoma and AMD have profound effect on the family members and other loved ones who provide most of the care for people having this disease. Unfortunately, the cure for glaucoma and AMD has not yet been discovered.
AMD is characterized by abnormal extracellular deposits, known as drusen, the hallmark sign of AMD, that accumulate along the basal surface of the retinal pigmented epithelium. Although drusen is common in older individuals, large numbers of drusen and/or extensive areas of confluent drusen represent a significant risk factor for AMD. Widespread drusen deposition is associated with retinal pigmented epithelial cell dysfunction and degeneration of the photoreceptor cells. See Johnson et al., Proc Natl Acad Sci USA 99, 11830 (2002). There are two types of AMD, dry and wet. The dry type of AMD is characterized by a geographic atrophy that progresses slowly over many years. In the wet type of AMD choroidal neovascularization occurs that result in a dense fibrovascular scar that may involve the entire macular area. The wet type of AMD is more sight threatening than the dry type and is responsible for 90% of cases of severe visual loss in elderly population. See Chopdar et al., BMJ 326, 485 (2003).
Glaucoma is a chronic neurodegeneration of the optic nerve, retinal ganglion cells, that result in irreversible vision loss. See Khaw et al., BMJ 320, 1619 (2000).
The pathogenesis of glaucoma and AMD has recently been linked to deposition of beta-amyloid (βA) in retinal cells of the eyes. It was recently demonstrated that retinal ganglion cell death in glaucoma involves βA neurotoxicity at the molecular level. See McKinnon et al., IOVS 43, 1077 (2002). βA was also shown to associate with a substructural vesicular component within drusen and was found to correlate with the location of degenerating photoreceptors and retinal pigmented epithelium cells. See Dentchev et al., Mol Vis 14, 184 (2003). βA deposition was found an important component of the local inflammatory events that contribute to atrophy of the retinal pigmented epithelium, drusen biogenesis and the pathogenesis of AMD. See Johnson et al., Proc Natl Acad Sci USA 99, 11830 (2002).
Thus, therapeutic approaches that can modulate βA toxicity have been hypothesized to represent important methods for controlling the onset of glaucoma and macular degeneration. It is envisioned that if retinal cells can be protected from βA-induced toxicity, the onset of glaucoma and AMD may be delayed or prevented.
Current glaucoma treatment focuses on lowering intraocular pressure, the major risk factor for the disease. Glaucoma has been treated medically, surgically, or with laser to lower intraocular pressure that can slow the disease progression. Pharmacological treatment approaches are: cholinergic agents (pilocarpine—increases outflow of the aqueous humour; beta blockers—reduce aqueous secretion), oral carbonic anhydrase inhibitors (acetazolamide and dorzolamide—reduces aqueous secretion), alpha-2 adrenergic agonists (apraclonidine and brimonidine), and prostaglandin agonists (latanoprost—opens up an alternative pathway for aqueous outflow by altering the resistance of the extracellular matrix). See Khaw et al., BMJ 320, 1619 (2000) and Khaw et al., BMJ 328, 156 (2004).
One current treatment approach for AMD is a technique called photodynamic therapy that uses verteporfin as the photosensitizer. Long term supplementation with high dose zinc and antioxidant vitamins (A, C, and E) showed a significant reduction in the relative risk of developing neovascular AMD. As a preventive measure against the disease progression and the onset of AMD, carotenoids lutein and zeaxanthin, which are potent antioxidants found in high concentrations in the macular retina are found to be effective. See. Chopdar et al., BMJ 326, 485 (2003).
One important pharmacological approach related to βA-induced neurodegenerative disease preventive and neuroprotective interventions may be antioxidant therapy. See Kumar et al., Int J Neurosci 79, 185 (1994), Lucca, et al., Brain Res 764, 293 (1997), Manelli et al., Brain Res Bull 38, 569 (1995), Parnetti et al., Drugs 53, 752 (1997), Preston et al., Neurosci Lett 242, 105 (1998), and Zhou, et al., J Neurochem 67, 1419 (1996). In designing inhibitors of βA toxicity, it was found that inducing changes in aggregation kinetics and in higher order structural characteristics of βA aggregate may prove to be effective in reducing βA toxicity. See Ghanta et al., J Biol Chem 271, 29525 (1996). Synthetic inhibitors that interact with βA was shown to completely block βA toxicity against PC12 cells, demonstrating that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity. See Yaar et al., J Clin Invest 100, 2333 (1997) and Hertel et al., Proc Natl Acad Sci USA 94, 9412 (1997). These results suggest that βA toxicity can be mediated through a physicochemical interaction with cell membranes.
There is strong interest in discovering potentially valuable natural sources for drug development. One reasonable source of such natural products involves medicinal plants that have been in use throughout history for treating various ailments. Thus, the discovery of potentially valuable plants that can protect neurons from βA insult is of interest.
Curcuma longa (Zingiberaceae) has been used as curry spice and a well known constituent of Indonesian traditional medicine. See Nurfina et al., Eur J Med Chem 32, 321 (1997). One of the important constituents of turmeric is curcumin that has been known as a natural antioxidant with antitumor activity. See Ruby et al., Cancer Lett 94, 79 (1995). From turmeric, curcuminoids with antioxidant property have been demonstrated to protect neuronal cells from βA insult. See Kim DSHL et al., Neurosci Lett 303, 57 and Park S Y et al., J Nat Prod 65, 1227 (2002). A representative list of Curcuma sp. include C. longa, C. aromatica, C. domestica, C. xanthorrhiza, and C. zedoaria. 
Zingiber officinale (Zingiberaceae) is one of the world's favorite spices, probably discovered in the tropics of Southeast Asia. Ginger has benefited humankind as a wonder drug since the beginning of recorded history. See Jitoe et al., J Agric Food Chem 40, 1337 (1992), Kikuzaki et al., J Food Sci 58, 1407 (1993) and Schulick, Herbal Free Press, Ltd. (1994). From ginger, shogaols with antioxidant property have also been demonstrated to protect neuronal cells from βA insult. See Kim et al., Planta Medica 68, 375 (2002). A representative list of Zingiber sp. include Z. officinale, Z. zerumbet, and Z. mioga. 
Ginkgo (Ginkgo biloba (Ginkgoaceae)) is an herbal that has been used to treat neurologic ailment for thousand years as an Asian traditional medicine. Ginkgo leaf extract has shown to exhibit potent antioxidant activity and are widely used in the dietary supplement industry. The antioxidant activity of ginkgo has shown to be primarily contributed by diterpenes such as ginkgolides, bilobilide, flavonoids, and ginkgolic acids. See Hopia et al., J Agric Food Chem 44, 2030 (1996) and Nakatani et al., Agric Biol Chem 47, 353 (1983).
Sage (Salvia officinalis L. (Lamiaceae)) and Rosemary (Rosmarinus officinalis L. (Labiatae)) are spices widely used for flavoring and seasoning foods. These spices have shown to contain potent diterpenoid antioxidants such as carnosic acid, carnosol, rosmarinic acid, rosmanol, epirosmanol, rosmadial, isorosmanol etc. See Haraguchi et al., Planta Med 61, 333 (1995). Inatani et al, Agric Biol Chem 47: 521 (1983). Nakatani et al., Agric Biol Chem 48: 2081 (1984). Inatani et al., Agric Biol Chem 46: 1661 (1982). Wang et al., J Agric Food Chem 46: 2509 (1998). Wang et al., JAgric Food Chem 46: 4869 (1998).